Synthesis, in vitro anticancer activity and SAR studies of arylated imidazo[1,2-a]pyrazine–coumarin hybrids

Abstract

A new series of imidazo[1,2-a]pyrazine–coumarin hybrids have been synthesized by the combination of two biologically active moieties, imidazo[1,2-a]pyrazine and coumarin, followed by the Suzuki–Miyaura cross coupling reaction for monoarylation at the C6 position and symmetrical/unsymmetrical diarylation at the C3 and C6 positions. These compounds were further screened for their in vitro antitumor activities.

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Heterocyclic moieties play an important role in pharmaceutical, veterinary and agrochemicals.¹ Among the various heterocycles, imidazo[1,2-a]pyrazine, a well known privileged fused heterocyclic motif, has shown its importance in the search for anticancer agents with variations at different positions of the core.² Imidazo[1,2-a]pyrazine exhibited prominent anticancer activity with the inhibition of Aurora kinase,³ phosphoinositide-3-kinase,⁴ breast tumor kinase/protein tyrosine kinase 6,⁵ check point kinase,⁶ spleen tyrosine kinase,⁷ topoisomerase-II⁸ and cyclin dependent kinase⁹ (Fig. 1). Coumarin is also a biologically active heterocyclic moiety possessing anticancer,¹⁰ anti HIV,¹¹ antituberculosis,¹² antihypercholesterolemic activities¹³ etc. Currently there is a focus on molecular hybridization to obtain a more active pharmacophore in a single biological molecule by joining two heterocyclic pharmacophores.¹⁴ The concept of hybridization is leading to a revolution in the field of drug design and development to obtain a hybrid drug with improved characteristics and minimal side effects as compared to the parent molecules. Therefore, hybrids of coumarin with various heterocyclic moieties viz., coumarin–benzimidazole,¹⁵ coumarin–benzothiazole¹⁶ and coumarin–chalcone¹⁷ (Fig. 2) have been reported in the literature, having great biological significance as anticancer agents.

Fig. 1 Imidazo[1,2-a]pyrazines as anticancer agents.

Fig. 2 Coumarin based hybrids as anticancer agents.

To the best of our knowledge, no reports on the anticancer activity of imidazo[1,2-a]pyrazine with a biologically active coumarin moiety have been present in the literature until now. In view of the previous rationale and in continuation of an ongoing program to find new structural leads with potential chemotherapeutic activities using molecular hybridization, in the present study, a new series of hybrids using imidazo[1,2-a]pyrazine and coumarin have been synthesized. These compounds have further been used for the Suzuki–Miyaura cross coupling reaction for monoarylation at the C6 position, and symmetrical/unsymmetrical diarylation at the C3 and C6 positions. The compounds were further screened for their in vitro anticancer activity on a panel of 60 human cancer cell lines viz., leukaemia, non small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. A molecular docking study has also been performed to support the effective binding of compounds at the active sites of enzymes.

2-Aminopyrazine 1 was treated with N-bromosuccinimide (NBS) in DMSO and water (9 : 1) at room temperature for 6 h to give 2-amino-3,5-dibromopyrazine 2 in 90% yield¹⁸ followed by cyclization with 50% aq. chloroacetaldehyde in isopropyl alcohol at 110 °C for 12 h to obtain 6,8-dibromoimidazo[1,2-a]pyrazine 3 (ref. 5) in 80% yield. Compound 3 was again brominated with NBS in acetonitrile (ACN) at room temperature for 2 h to afford 3,6,8-tribromoimidazo[1,2-a]pyrazine 4 (ref. 19) in 90% yield. 3,6,8-Tribromoimidazo[1,2-a]pyrazine 4 was then stirred with 7-hydroxy-4-methylcoumarin 5 in the presence of K₂CO₃ and DMF at room temperature for 12 h to afford 7′-(3,6-dibromoimidazo[1,2-a]pyrazin-8-yloxy)-4′-methyl-2H-chromen-2′-one 6 in 85% yield (Scheme 1).

Scheme 1 Synthesis of the imidazo[1,2-a]pyrazine–coumarin hybrid.

Palladium catalyzed Suzuki–Miyaura cross coupling of compound 6 with 2-methoxyphenyl boronic acid (1 eq.) in the presence of K₂CO₃ (1 eq.) and 5 mol% of Pd(PPh₃)₄ in DME : H₂O (9 : 1) under an inert atmosphere afforded C6-monoarylated 7a and C3, C6 diarylated 7b products in 61% and 33% yields, respectively (Table 1, entry 1). Similarly, compound 6 was also treated with 2-furan boronic acid under the same reaction conditions to give compounds 8a and 8b in 48% and 25% yields, respectively (Table 1, entry 2). Reaction of compound 6 with 4-chlorophenyl boronic acid afforded the C6 monoarylated product 9a and the C3, C6 diarylated product 9b in 40% and 42% GCMS yields, respectively. These two compounds could not be separated through column chromatography and were used as such without purification (Table 1, entry 3). However, reactions of 6 with other aryl boronic acids under the same reaction conditions gave only C3, C6 diarylated products 10b–19b with 54–74% yields (Table 1, entries 4–13). Monoarylated products with these boronic acids could not be separated as these were formed only in trace amounts <5% (determined by GC-MS). Reaction of compound 6 with naphthalene-1-boronic acid was not successful. This might be due to steric crowding of the bulky naphthyl ring.

Table 1 Reactions of 3,6-dibromoimidazo[1,2-a]pyrazine–coumarin hybrid 6 with aryl boronic acids

Entry	Time h	R1B(OH)2	Products^a (%)
a Isolated yields.b GC-MS yields (<5%).
1	8		7a (61)	7b (33)
2	11		8a (48)	8b (25)
3	10		9a (40)^b	9b (42)
4	10		^—	10b (66)
5	9		^—	11b (74)
6	12		^—	12b (70)
7	12		^—	13b (69)
8	9		^—	14b (72)
9	12		^—	15b (60)
10	8		^—	16b (73)
11	10		^—	17b (70)
12	11		^—	18b (68)
13	12		^—	19b (54)

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Subsequently, the use of the monoarylated product was implemented for unsymmetrical diarylation via a cross coupling reaction at the C3 position. C6 Monoarylated imidazo[1,2-a]pyrazine–coumarin hybrid 7a was further refluxed with a variety of aryl boronic acids (1.0 eq.) in the presence of 5 mol% of Pd(PPh₃)₄, K₂CO₃ (1.1 eq.) in DME : H₂O (9 : 1) for 6–8 h to give C3, C6 unsymmetrical diarylated imidazo[1,2-a]pyrazine–coumarin hybrids 20–29 in 52–75% yields (Table 2). However, the Suzuki reaction of a mixture of monoarylated and symmetrical diarylated compounds (9a and 9b) with 2-thiophene boronic acid under the same reaction conditions and after column chromatography afforded C3, C6 symmetrical and unsymmetrical disubstituted imidazo[1,2-a]pyrazine–coumarin hybrids 9b and 30 in 42% and 61% isolated yields, respectively. All the synthesized compounds have been well characterized by ¹H and ¹³C NMR as well as mass spectrometry (ESI†).

Table 2 Reactions of monoarylated-3-bromoimidazo[1,2-a]pyrazine–coumarin hybrids 7a and 9a with different aryl boronic acids

Entry	Starting material	R2B(OH)2	Product	Time (h)	Yield^a (%)	Mp (°C)
a Isolated yields.
1	7a			6	20 (64)	221–223
2	7a			7	21 (52)	204–205
3	7a			6	22 (69)	194–195
4	7a			8	23 (72)	202–203
5	7a			8	24 (55)	179–180
6	7a			7	25 (75)	235–237
7	7a			7	26 (65)	243–245
8	7a			8	27 (55)	204–206
9	7a			7	28 (64)	239–241
10	7a			8	29 (53)	>300
11	9a			8	30 (61)	187–189

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The assigned regiochemistry of monoarylation at the C6 position and unsymmetrical diarylation at the C3 and C6 positions of the imidazo[1,2-a]pyrazine–coumarin hybrid was confirmed by considering 2D NOE difference experiments (ESI, Fig. S7 and S77†). The singlet of C5H of imidazo[1,2-a]pyrazine 7a showed a positive NOE signal with protons of the methoxy group of the 2-methoxyphenyl ring while negative NOE signal of the singlet of C2H of imidazo[1,2-a]pyrazine with protons of the methoxy group of the 2-methoxyphenyl ring was observed, indicating the monoarylation primarily at the C6 position (Fig. 3). On the other hand, singlets of C2H and C5H of imidazo[1,2-a]pyrazine ring in compound 28 showed positive NOE signals with protons of C2′′′ and C6′′′ of the 4-methoxyphenyl ring present at the C3 position. The negative NOE signal of the singlet of C2H at imidazo[1,2-a]pyrazine with the methoxy protons of the 2-methoxy phenyl ring (at the C6 position) and the positive NOE signal with the 4-methoxy phenyl ring (at the C3 position) confirmed the second arylation at the C3 position of imidazo[1,2-a]pyrazine 28 (Fig. 4).

Fig. 3 2D NOEs ¹H, ¹H correlations used for structural assignment of compound 7a.

Fig. 4 2D NOEs ¹H, ¹H correlations used for structural assignment of compound 28.

Compounds 6, 7a, 9b, 10b, 11b, 16b, 28 and 30 were submitted to NCI, USA to test their in vitro anticancer activities against a panel of 60 human cancer cell lines at a single dose concentration of 10 μM.^20–23 Compound 6 with bromo at the C3 and C6 positions showed growth inhibition against the renal cancer cell line 786-0 with a GI value of 71.02% and breast cancer cell lines MCF-7 and MDA-MB-231/ATCC with respective GI values of 77.75% and 70.43%. But monoarylation with 2-methoxy phenyl at the C6 position 7a displayed poor anticancer activity. Amongst the symmetrical diarylated compounds, 4-chlorophenyl rings at both the C3 and C6 positions 9b showed no significant growth inhibition against cancer cell lines while 2-thiophene moieties at the C3 and C6 positions 10b showed excellent inhibition against non small cell lung cancer cell line HOP-92 with a GI value of 92.29%. Replacement with six membered phenyl rings at the C3 and C6 positions 11b exhibited excellent anticancer activity against the renal cancer cell line A-498 with a GI value of 90.12% while moderate activity against renal cancer RXF-393 and non small cell lung cancer cell line HOP-92 with respective GI values of 71.67% and 77.65% was observed. Substitution with 4-methoxy groups at phenyl rings at the C3 and C6 positions of imidazo[1,2-a]pyrazine 16b displayed a broad spectrum of anticancer activity towards various cancer cell lines viz., breast cancer cell line MDA-MB-231/ATCC, melanoma cell line LOX IMVI, CNS cancer cell line SNB-75, non small cell lung cancer cell line A549/ATCC and prostate cancer cell line PC-3 with GI values of 93.71%, 91.98%, 85.69%, 83.52% and 82.27%, respectively. It also displayed good inhibition with other cancer cell lines viz., non small cell lung cancer cell line NCI-H460 (GI: 73.64%), colon cancer cell line HT29 (GI: 74.56%), CNS cancer cell lines U251 (GI: 77.45%) and SF-295 (GI: 76.68%), melanoma cancer cell lines MALME-3M (GI: 73.25%), SK-MEL-5 (GI: 71.37%) and UACC-257 (GI: 70.84%), ovarian cancer cell lines OVCAR-8 (GI: 73.68%) and NCI/ADR-RES (GI: 70.09%), renal cancer cell line SN12C (GI: 75.49%), prostate cancer cell line DU-145 (GI: 70.53%) and breast cancer cell line BT-549 (GI: 70.80%) (Fig. 5). Compound 16b exhibited more than 70% growth inhibition for most of the tumor cell lines and was much better than 5-fluorouracil (positive control) in tested derivatives. It showed higher activity than 5-fluorouracil (5-FU) in non-small cell lung cancer cells (A549/ATCC, HOP-62, NCI-H23 and NCI-H460), colon cancer cells (HCT-116 and HT-29), melanoma (LOX IMVI, MALME-3M, MDA-MB-435, SK-MEL-5 and UACC-257), prostate cancer (PC-3 and DU-145) and breast cancer cells (MCF-7, MDA-MB-231/ATCC and BT-549). In the series of unsymmetrical compounds, imidazo[1,2-a]pyrazine with 4-methoxy phenyl at the C3 and 2-methoxy phenyl at the C6 position 28 displayed poor anticancer activity while substitution with 2-thiophene at the C3 position and 4-chlorophenyl at the C6 position 30, increases the activity with more selectivity towards the melanoma cancer cell line MALME-3M with a GI value of 77.82% (Table 3).

Fig. 5 The percentage growth inhibition of compound 16b over the full panel of tumor cell lines.

Table 3 Percentage (%) growth inhibition (GI) of compounds 6, 7a, 9b, 10b, 11b, 16b, 28, 30 and 5-FU over the full panel of 60 tumor cell lines at a concentration of 10 μM^a

Cell line type	Cell line name	6	7a	9b	10b	11b	16b	28	30	5-FU
a NT: not tested; L: lethal.b 50–60% growth inhibition.c 60–70% growth inhibition.d 70–90% growth inhibition.e 90–100% growth inhibition.f Highly potent compounds.
Leukemia	CCRF-CEM	—	—	19.41	—	22.39	—	—	14.72	57.13
	HL-60(TB)	69.19^c	—	13.22	32.40	14.82	NT	—	32.62	47.90
	K-562	—	—	14.92	18.35	38.24	NT	—	47.06	42.38
	MOLT-4	12.54	—	25.52	21.25	41.55	NT	—	20.34	43.13
	RPMI-8226	—	—	—	29.69	48.96	36.31	—	17.02	41.41
	SR	30.60	—	49.13	54.39^b	45.46	NT	—	27.81	24.82
Non-small cell lung cancer	A549/ATCC	22.21	13.25	—	41.45	43.86	83.52^d	—	—	34.25
	EKVX	24.43	24.04	—	35.95	33.20	49.84	—	—	58.40
	HOP-62	48.29	22.74	—	40.98	31.25	59.14^b	—	—	47.89
	HOP-92	62.81^c	—	—	92.29^e	77.65^d	−13.56^f	11.79	15.61	50.64
	NCI–H226	46.40	27.46	—	16.99	55.35^b	29.28	—	—	69.55
	NCI–H23	36.05	25.40	21.91	43.37	37.56	66.97^c	—	28.24	39.01
	NCI–H322M	23.24	18.07	—	10.57	14.03	18.02	—	11.91	59.50
	NCI–H460	26.12	—	—	49.53	36.05	73.64^d	—	—	13.07
	NCI–H522	45.23	36.87	—	25.35	52.85^b	60.39^c	—	—	58.02
Colon cancer	COLO 205	—	—	—	25.05	29.50	43.89	—	11.30	40.22
	HCC-2998	—	11.61	—	21.86	18.95	22.91	—	—	L^f
	HCT-116	44.49	20.47	—	41.34	60.66^c	66.14^c	—	18.23	17.83
	HCT-15	—	—	36.21	27.11	31.38	36.61	—	29.83	26.56
	HT29	—	—	52.34	53.36^b	46.79	74.56^d	—	49.09	27.19
	KM12	21.08	—	18.81	40.37	24.49	57.01^b	—	23.63	40.70
	SW-620	—	—	33.27	43.33	11.72	60.98^c	—	13.73	50.12
CNS cancer	SF-268	43.44	22.13	—	23.75	35.64	34.29	—	14.33	59.05
	SF-295	—	11.75	—	52.52^b	45.77	76.68^d	—	—	69.16
	SF-539	52.78^b	18.31	—	40.04	45.41	−21.15^f	—	—	L^f
	SNB-19	25.37	—	NT	NT	35.56	NT	NT	—	65.96
	SNB-75	−2.07^f	68.32	—	64.44^c	−1.39^f	85.69^d	15.18	—	65.93
	U251	52.35^b	17.29	11.95	52.46^b	32.65	77.45^d	—	19.30	50.35
Melanoma	LOX IMVI	25.67	24.60	15.63	61.63^c	43.60	91.98^e	—	21.46	30.40
	MALME-3M	53.11^b	24.30	56.42^b	60.88^c	−9.77^f	73.25^d	—	77.82^d	58.21
	M14	17.22	—	35.77	32.45	32.28	61.41^c	—	61.10^c	NT
	MDA-MB-435	12.82	—	24.52	38.65	27.95	62.77^c	—	20.55	36.66
	SK-MEL-2	22.00	—	—	31.03	48.10	50.58^b	—	—	95.52
	SK-MEL-28	—	—	61.69^c	76.09^d	32.37	−17.37^f	—	40.28	NT
	SK-MEL-5	59.65^b	13.41	15.59	57.50^b	61.69^c	71.37^d	—	30.47	33.75
	UACC-257	10.94	—	11.68	51.80^b	34.28	70.84^d	—	—	19.56
	UACC-62	14.79	—	—	28.89	18.62	49.89	—	—	39.77
Ovarian cancer	IGROV1	22.57	26.28	—	20.77	16.32	29.38	—	—	51.29
	OVCAR-3	43.29	13.76	—	32.04	23.36	53.41^b	—	—	47.41
	OVCAR-4	54.55^b	—	14.41	45.94	39.32	−18.64^f	—	25.03	59.40
	OVCAR-5	—	—	—	—	—	12.00	—	—	44.34
	OVCAR-8	35.35	—	—	47.45	38.43	73.68^d	—	—	NT
	NCI/ADR-RES	42.47	22.95	21.20	46.63	54.62^b	70.09^d	—	12.56	47.65
	SK-OV-3	14.52	13.64	—	—	36.88	—	—	—	77.56
Renal cancer	786-0	71.02^d	—	NT	NT	32.72	NT	—	—	48.79
	A498	—	—	—	35.60	90.12^e	29.38	—	—	L^f
	ACHN	34.24	22.25	—	42.72	51.39^b	53.41^b	—	—	39.31
	CAKI-1	—	10.67	—	32.18	14.82	28.33	—	—	39.40
	RXF 393	55.79^b	11.01	29.51	36.41	71.67^d	−20.76^f	—	12.23	34.33
	SN12C	24.19	—	—	26.17	32.41	75.49^d	—	—	54.04
	TK-10	—	—	—	46.11	19.47	64.26^c	—	—	66.98
	UO-31	41.18	37.92	14.45	39.73	59.18^b	25.03	18.30	31.13	41.30
Prostate cancer	PC-3	17.90	23.95	12.06	47.04	60.82^c	82.27^d	—	19.78	58.26
	DU-145	24.95	12.94	30.37	44.61	31.96	70.53^d	—	18.46	35.52
Breast cancer	MCF7	77.75^d	24.89	26.92	40.08	49.54	55.81^b	—	44.11	11.55
	MDA-MB-231/ATCC	70.43^d	31.76	26.69	36.78	49.62	93.71^e	24.30	—	78.17
	HS 578T	30.14	15.90	12.25	32.35	41.43	42.41	—	—	L^f
	BT-549	—	—	10.59	75.01^d	17.35	70.80^d	—	—	37.81
	T-47D	46.34	22.05	—	45.27	61.52^c	54.65^b	—	15.70	56.78
	MDA-MB-468	58.60^b	30.06	15.17	45.34	55.77^b	56.11^b	—	17.46	NT

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Compound 16b was also evaluated for toxicity to Hek293 (human embryonic kidney) cell lines using the MTT assay.²⁴ It was observed that compound 16b showed only 17%, 15%, 9%, 5% and 3% cytotoxicity to Hek293 cells at 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M concentrations, respectively (Fig. S84, ESI†). The compound showed only 17% of toxicity to Hek293 cells even at 100 μM concentration. These data indicated that compound 16b showed potent anticancer activity and low toxicity to normal cells.

The partition coefficient of the compounds was also studied in an octanol/water system for the determination of log P values by a shake-flask method²⁵ (ESI†). It was observed that compounds 11b and 16b showed higher log P values (Table 4), which suggested that the higher antitumour activity of these compounds was related to the lipophilicity. Thus, lipophilicity is a crucial factor for the activity of the synthesized compounds.

Table 4 Experimentally determined lipophilicity^a

Compounds	P	log P
a P—partition coefficient; log P—logarithm of the partition coefficient.
6	239.88	2.38
7a	489.77	2.69
9b	371.53	2.57
10b	467.73	2.67
11b	2511.88	3.40
16b	758.57	2.88
28	257.03	2.41
30	380.19	2.58

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Structure–activity correlation, based on the number of cancer cell lines that were inhibited by each compound revealed that the nature of the substituents at the C3- and C6-positions of imidazo[1,2-a]pyrazine affected the biological activity. Compounds 10b, 11b and 16b showed comparatively higher activity than 6, 7a, 9b, 28 and 30, suggesting that there is much difference in antitumor activity with different substitutions of phenyl rings. The antitumor results indicated that compound 6 with a coumarin moiety at the C8 position and bromo at the C3 and C6 positions displayed only moderate anticancer activity. The anticancer activity has been slightly increased with the substitution of 2-thiophene rings at the C3 and C6 positions, as in the case of compound 10b. Higher activity has been achieved with substitution of phenyl rings 11b and 4-methoxyphenyl rings 16b at the C3 and C6 positions. On the other hand, substitution with 4-chlorophenyl at the C6 position and 2-thiophene at the C3 position as in case of compound 30 decreased the activity. It has been revealed that symmetrical diarylated imidazo[1,2-a]pyrazine–coumarin hybrids 11b and 16b showed higher activity than unsymmetrical diarylated hybrids 28 and 30. These studies indicated that substitution of the coumarin heterocycle with various phenyl derivatives on imidazo[1,2-a]pyrazine gave highly potent anticancer activities towards 60 human cancer cell lines. Overall, 16b has been found to be the most effective member of this series of compounds and showed a broad spectrum of activity against melanoma cancer cell lines.

Preliminary anticancer screening showed that compound 16b has been found to be the most active member of the imidazo[1,2-a]pyrazine–coumarin hybrids and showed excellent inhibition against melanoma over other cancer cell lines. So, in order to observe the molecular interactions of the compound in the active site of an enzyme for melanoma cancer, a docking experiment was performed. The crystal coordinates of an enzyme used in melanoma were downloaded from a protein data bank (https://www.rcsb.org)²⁶ (PDB code: 3OG7).²⁷ Compound 16b showed H-bonding interactions of the N1 (d = 2.93 Å), N4 (d = 2.72 Å) and N7 (d = 1.75 Å) atoms of imidazo[1,2-a]pyrazine with the G518 amino acid residue of the coil of the enzyme. The N7 atom of the pyrazine ring of imidazo[1,2-a]pyrazine also showed H-bonding interaction with the M517 (d = 2.17 Å) amino acid residue of the enzyme. The oxygen atom of the 4-methoxy phenyl at the C6 position of imidazo[1,2-a]pyrazine showed H-bonding interactions with the R781 amino acid residue of the enzyme (d = 2.85 Å). The carbonyl group of the coumarin ring interacts with the W531 amino acid residue of the β-strand of the enzyme with d = 2.56 Å. The oxygen atom and carbonyl group of the coumarin ring also showed H-bonding interactions with Q530 amino acid residues of the β-strand of the enzyme (d = 1.77 Å and d = 2.31 Å). Therefore, docking of compound 16b in the active site of this enzyme indicated the probable mode of action for its anticancer activity (Fig. 6).

Fig. 6 Docking of compound 16b in the active site of 3OG7. H-bonds of compound 16b with different amino acid residues are visible. Carbon atoms are given in green colour.

Conclusion

In summary, an imidazo[1,2-a]pyrazine–coumarin hybrid has been synthesized by a nucleophilic substitution approach at the C8 position from the easily accessible 3,6,8-tribromoimidazo[1,2-a]pyrazine and 7-hydroxy-4-methyl coumarin. This compound was further functionalized at the C6 position for monoarylation and the C3, C6 positions for symmetrical diarylations using palladium catalyzed C–C coupling. Subsequent use of monoarylated products has been implemented for the synthesis of unsymmetrical diarylated imidazo[1,2-a]pyrazine–coumarin hybrids in moderate to good yields. Evaluation of selected compounds for anticancer activity revealed that the symmetrical diarylated hybrids 11b and 16b showed a broad spectrum of anticancer activity towards most of the cancer cell lines. These compounds also have good lipophilicity that qualifies them to have good pharmacokinetics and drug bioavailability. A molecular docking study also further supported the inhibitory activity of 16b and helped in understanding the interactions between the ligand and enzyme active sites. Further optimizations of the anticancer activity and pharmacokinetic profiling of these series of compounds are currently ongoing.

Acknowledgements

KP wishes to thank CSIR, New Delhi (02(0034)/11/EMR-II) for providing funds. RG is indebted to CSIR for SRF (09/677(0020)/2013.EMR-I). We wish to thank SAI Labs, Thapar University and Punjab University, Chandigarh for carrying out NMR and Mass analysis respectively. National Cancer Institute, USA and Institute for Industrial Research and Toxicology, India are greatly acknowledged for activities.

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Footnote

† Electronic supplementary information (ESI) available: Experimental section, ¹H, ¹³C NMR and mass spectra of all new compounds 6, 7a–8a, 7b–19b, 20–30, antitumor methodology and activities of all selected compounds 6, 7a, 9b, 10b, 11b, 16b, 28 and 30. See DOI: 10.1039/c5ra00584a

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